1. Field of Invention
The present invention concerns a simple, robust and precise instrument for constructing tissue arrays. The instrument may be operated manually or automatically.
2. Discussion of the Related Art
Some cancer patients respond well to a particular cancer therapy or combination of therapies. Others do not, but may respond to a different treatment. Scientists at the National Human Genome Research Institute (NHGRI) at the National Institutes of Health (NIH), in collaboration with the University of Tampere in Finland and the University of Basel in Switzerland, are developing a new research tool, which they call the "tissue chip," that they expect will eventually help them learn how to distinguish among subgroups of cancer patients and eventually predict which subgroups will respond to specific therapies. The tissue chip technology, they believe, will also help illuminate the process of cancer development. Such detailed new information can then be used to identify critical molecules for development of cancer therapies.
The tissue chip is a thin section of a tissue microarray that permits massive parallel processing of biological samples, making it possible for researchers to simultaneously compare a variety of molecular markers--DNA, RNA, and protein--in cancer tissues from hundreds or thousands of patients. As many as 1,000 tissue biopsies from individual tumors can be studied in a single tumor tissue microarray. The tissue chip thus makes it possible to simultaneously test thousands of patient tissue specimens which pathology laboratories have traditionally analyzed one specimen at a time. The power of this technology is expected to accelerate numerous areas of research, including testing of newly isolated genes to determine if they may be of clinical utility as molecular cancer markers.
In a study of breast cancer tissue microarrays (Kononen et al "Tissue microarrays for high-throughput molecular profiling of tumor specimens", Nature Medicine Vol. 4, Number 7 July 1998 pp. 844-847) researchers analyzed six gene amplifications and expression of the p53 and estrogen receptor genes believed to play a role in breast cancer. These researchers used genotyping in the search for cancer susceptibility loci, comparative genomic hybridization (CGH) for copy number alterations, as well as cDNA microarray technology for gene expression surveys, and recently discovered amplification of a steroid receptor co-activator, AIB1, in breast cancers as well as amplification of the androgen receptor (AR) gene in recurrent, hormone-refractory prostate cancers.
Each microarray is a block which may be comprised of 1,000 individual cylindrical tissue biopsies or "cores". Each microarray can be sliced into 200 consecutive sections of 5 micrometers each by traditional means (i.e., microtomes, etc.). The result is multiple nearly identical sections (tissue chips), each of which is used to make one ordinary microscope slide. With each of the cores then being represented as a minuscule dot in the same position in the matrix on each of the 200 microscope slides, it became possible to quickly analyze hundreds of molecular markers in the same set of specimens. Sections of the microarray provide targets for parallel in situ detection of DNA, RNA and protein targets in each specimen on the array, and consecutive sections allow the rapid analysis of hundreds of molecular markers in the same set of specimens. In the Kononen et al study, the tissue chip made it possible to complete in about one week what traditional methods would have taken from 65 to 12-months.
The tissue chip is also expected to be particularly useful in analyzing the thousands of tumor tissue samples stored in pathology labs all over the world. Previously, it would not have been considered practical to analyze these thousands of archived tumor tissue samples for hundreds of molecular markers--one at a time. Now, with the tissue chip, pathologists can take their existing archives, turn them into tumor arrays, and analyze an entire archive with just a few experiments. Pathologists can also array archived tissue samples from clinical trials of existing cancer drugs, and look for markers--a gene expression pattern or set of genetic changes in the tissue--associated with whether or not a specific participant in the trial responded to the therapy.
While tissue chips may significantly accelerate the assaying process, it has created a new challenge--a considerable investment in time and labor is necessary to manually extract samples from donor tissue and to assemble these specimens into a tissue array (Battifora, H., "The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing", Laboratory Investigation Vol. 55, pp. 244-248, 1986).
U.S. Pat. No. 4,820,504 entitled "Multi-specimen tissue blocks and slides" (Battifora) teaches a method of preparing a multi-specimen tissue block, and sections thereof, comprising forming a plurality of different antigenically reactive tissue specimens into rods having a relatively small cross-sectional area and a relatively great length, bundling the rods in a substantially parallel relationship on a casing, wrapping the rods in the casing, embedding the wrapped rods in an embedding medium to form a tissue block in which the rods are perpendicular to the face of the block, and dividing the block into sections which each contain a cross-section of each of the rods. While many specimens could be located in a compact area, it became difficult or impossible to track the identity of the various specimens.
U.S. Pat. No. 5,002,377 entitled "Multi-specimen slides for immunohistologic procedures" (Battifora) addresses this identity problem and teaches a process for producing a slide bearing a spaced array of specimen fragments which comprises (i) cutting at least one specimen into a plurality of narrow strips; (ii) separating the plurality into groups of specimen strips; (iii) separately positioning strips from the groups in parallel grooves in a mold; (iv) embedding the strips in the mold in a first embedding medium to provide a structure comprising a base member having opposed first and second surfaces, the first surface being substantially planar; the second surface having ridges containing a specimen strip extending therefrom; (v) forming a stack of elements, each element corresponding to the structure, with the terminal surface of the ridges of an upper structure abutting the substantially planar first surface of the next lower structure; the spaces between the ridges defining channels for receipt of a fluid; (vi) embedding the stack in a second embedding medium to form a block having a spaced array of parallel specimen strips embedded therein; the strips being so arranged that a section of the block includes a spaced array of cross-sections of each of the embedded specimen strips; (vii) dividing the block into sections each containing a spaced array of cross-sections of each of the embedded specimen strips; (viii) mounting at least one of such block sections on a slide. While this method forms tissue samples into a grid pattern in which it is possible to track the identities of individual samples, the method is time consuming. Further, the method is not suitable for assembling into a single array hundreds of core samples from hundreds of individual donors.
More recently a technique has been developed wherein biological tissue arrays are constructed simply as arrays (rows and columns) of cores of biological tissue, each core having been punched from an individual donor tissue sample and embedded at a specific grid coordinate location in a sectionable block typically made of the same embedding material used for the donor tissue. The process of constructing micro-arrays involves two hollow needle-like punches. One, the "recipient punch", is slightly smaller and is used to create a hole in a recipient block, typically paraffin or other embedding medium. The other, the "donor punch", is larger and is used to obtain a core sample from a donor block of embedded biological tissue of interest. The punches are sized such that the sample obtained from the donor block (and corresponding to the inner diameter of the donor punch) just fits in the hole created in the recipient block (and corresponding to the external diameter of the recipient punch). Thus the sample snugly fits in the recipient block, and a precise array can be created. Either the donor or recipient block may be removed and be replaced, as desired, by one or more other donor or recipient blocks during the process to create a multi-specimen array. Micrometer drives or other precision linear positioning means are used to position the punch assembly with respect to the recipient block or the recipient block with respect to the punch assembly.
While it is possible, with time, patience, and skill, to create the above described tissue array using the instruments presently available, there is a clear need for improvement. Using slides and drive mechanisms to first move the recipient punch into position and alternatively, the donor punch, is cumbersome, expensive, slow and prone to misalignment errors. It is clearly desirable that the donor punch reach exactly the same position that the recipient punch reaches on the recipient block for a given setting of the micrometer drives. If it does not, the sample retrieved from the donor block will not pass smoothly into the hole just created for it in the recipient block, but instead will be damaged or lost.
The manual methods have largely been superceded by those aided by instruments in view of the speed, precision and increased pattern density of the latter. At least one semiautomatic system has been proposed but not realized. This semiautomatic system includes a punch platform mounted to move up and down (z-axis). A stylet and stylet drive are located centrally on the punch platform. On one side of the stylet there is provided an inclined recipient punch drive comprising a reciprocating ram that carries a tubular recipient punch at its distal end. On the other side of the stylet there is provided an inclined donor punch drive comprising a reciprocating ram that carries a tubular donor punch at its distal end.
To operate, first a tissue array block is placed below the punch platform, the recipient punch is extended until the recipient punch is below the stylet, and the punch platform (including the extended recipient punch, the retracted stylet, and the retracted donor punch) is lowered to cut a recipient core into the paraffin tissue array block. The punch platform is raised, a discard container is placed below the extended recipient punch, and the stylet is extended downwards into the recipient punch to expel the paraffin from the bore. The stylet is then retracted, and then the recipient punch is retracted. Next, a donor block is placed below the punch apparatus and the donor punch is extended until it occupies the space previously occupied by the recipient punch. The punch apparatus is lowered and the donor punch cuts a core sample from the donor block. The punch apparatus is then raised, and the recipient block is placed below the punch apparatus. The punch apparatus is lowered until the donor punch is located over the empty recipient hole, and the stylet is extended into the donor punch to expel the core sample into the recipient hole. The procedure is continued hundreds of times to form a tissue array block.
However, a number of disadvantages are associated with this apparatus. First, since there is only one stylet, and since the outer diameter of the stylet is dimensioned to fit snuggly within the inner diameter of the punch, it is only possible to use two punches having the same internal (and thus external), diameters in this instrument. Since needle-like donor and recipient punches are usually of different sizes, this instrument is not suitable for making micro-arrays. Second, considering that the steps for drilling and planting each core may have to be repeated 1,000 times to make a tissue array, and considering that each step introduces the possibility of operator error, there is a need to reduce the number of steps. Third, the fact that a single stylet is associated with two different punches makes it imperative that the punches, when extended, are positioned precisely below the stylet, as well as precisely above the target position on the donor or recipient block. The fact that the punches, when under the stylet, are in their fully extended position, means that the punches are in their structurally weakest position, and further, considering that any play or misalignment is amplified by the length of extension, any imprecision in positioning is magnified. Any misalignment of the punch could result in damage to the stylet and/or prevent proper planting of the donor core sample in the recipient block. Yet another deficiency is the inability to adjust the positioning of the punches with respect to the stylet. Further yet, this apparatus requires three actuator means--one for extending and retracting the stylet, one for the recipient punch, and one for the donor punch. As the number of moving parts increases, so does the likelihood of equipment failure. Finally, operation of the punch is not ergonomically or intuitively logical, thus increasing the likelihood of operator errors.
It is thus an object of the invention to provide an apparatus with which the precise sequential positioning of multiple punches can be effected reliably and inexpensively. It is a further object that this punch positioning as well as the punch stroke motion be easy to actuate by hand in a manually operated instrument. It is a further object of the invention to provide an instrument for the semi-automatic or automatic production of tissue arrays.
It is a further object of the present invention to overcome the cumbersome quality and imprecision of the prior art and to provide a simple and precise means of alternately positioning the two needle-like punches in a tissue micro-array constructing instrument.